GSM7892800: Anterior part, stage 45, rep2; Eptatretus burgeri; ATAC-seq
Hagfish embryos, at stages Dean 45 and 53, were divided into two parts, anterior and posterior halves, at the level of the somite pair 27. Nuclei were extracted separately from each part of embryos using 2 mL glass bouncers in ice-cold PBS and lysed in cold lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% Igepal). Each pool was further divided into two to obtain data from technical replicates. Transposition reaction was done on ~50,000 nuclei per sample and per replicate (total of 200,000 cells per embryo), with Tn5 enzyme (Illumina Nextera DNA Library Prep Kit), during 5 min at 37℃. DNA was purified using a QIAGEN MinElute kit following manufacturer instructions. Number of PCR cycles per sample were first determined as previously described (Buenrostro et al., 2013) using 1 uL of the Tn5'ed chromatin, and then a final PCR reaction with the rest of the chromatin was performed for each sample, with the following number of cycles: 7 for the embryo at stage 45, and 8 cycles for the embryo at stage 53. PCRs were performed with the NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs), using different sets of primers to multiplex the libraries. The resulting multiplexed libraries were checked in an Agilent 2100 BioAnalyzer using High Sensitivity DNA chips.